Abstract：
Both Ca2+ and protein kinase A (PKA) are multifaceted and ubiquitous signaling molecules, essential for regulating the intricate network of signaling pathways. However, their dynamics within specialized membrane regions are still not well characterized. By using genetically encoded fluorescent indicators specifically targeted to distinct plasma membrane microdomains, we have established a protocol that permits observing Ca2+/PKA dynamics in discrete neuronal microdomains with high spatial and temporal resolution. The approach employs a fluorescence microscope with a sensitive camera and a dedicated CFP/YFP/mCherry filter set, enabling the simultaneous detection of donor-acceptor emission and red fluorescence signal. In this detailed step-by-step guide, we outline the experimental procedure, including isolation of rat primary neurons and their transfection with biosensors targeted to lipid rafts or non-raft regions of plasma membrane. We provide information on the necessary equipment and imaging setup required for recording, along with highlighting critical parameters and troubleshooting guidelines for real-time measurements. Finally, we provide examples of the observed Ca2+ and PKA changes in specific cellular compartments. The application of this technique may have significant implications for studying cross-talk between second messengers and their alterations in various pathological conditions. © 2024 Wiley Periodicals LLC.
摘要：
Ca2+和蛋白激酶A（PKA）都是多方面和普遍存在的信号分子，对于调节复杂的信号通路网络至关重要。然而,它们在专门的膜区域内的动力学仍未得到很好的表征。通过使用基因编码的荧光指标，专门针对不同的质膜微域，我们已经建立了一个协议，允许在具有高空间和时间分辨率的离散神经元微域中观察Ca2/PKA动力学。该方法采用了带有灵敏相机和专用CFP/YFP/mCherry滤光片的荧光显微镜，能够同时检测供体-受体发射和红色荧光信号。在这个详细的分步指南中，我们概述了实验过程，包括分离大鼠原代神经元，并用靶向质膜脂质筏或非筏区域的生物传感器进行转染。我们提供有关记录所需的必要设备和成像设置的信息，以及突出显示实时测量的关键参数和故障排除指南。最后,我们提供了在特定细胞区室中观察到的Ca2和PKA变化的示例。该技术的应用可能对研究第二信使之间的串扰及其在各种病理条件下的变化具有重要意义。©2024Wiley期刊有限责任公司。
